This invention relates to the isolation of antigenic material from Schistosomes useful in the treatment of schistosomiasis in animals. More particularly, this invention relates to the extraction and isolation of antigenic material from living, adult schistosomes and the treatment of animals with the extracted antigenic material to treat and/or immunize against schistosome infection.
As used in this specification, the word "schistosome" will be used as a general term which will be understood to refer to the Trematode parasitic worms Schistosoma mansoni, Schistosoma japonicum, and Schistosoma hematobium.
Schistosomes are parasitic worms existing in many areas of the world, principally in Africa, Asia, and South America which, in their adult stage, are capable of residing in various organs of animals, particularly mammals, such as cattle and humans, primarily in the mesenteric and pelvic veins. The existance of schistosomes is particularly prevalent in tropical and underdeveloped countries of the world. Schistosoma have been characterized as the major helminth pathogen of man.
Schistosome eggs are produced by adult pairs of schistosomes located within the body of a host. The eggs are eliminated from the host's body, usually in the feces or urine. Schistosome eggs hatch into miracidia which develop into a larval stage in the body of any of several mollusks, generally of the snail type, as is well known by those skilled in the art. The larvae (cercaria) are then released to and live in open water systems, such as ponds, rivers, streams, etc. until they are able to infest an animal host.
The cercaria usually enter a mammalian host through the skin, generally in the extremities, such as a foot pad. The cercaria bore very quickly into the skin and travel through the lymphatic system of the host. In the lungs of the host, the cercaria develop to a second larval stage and move from the lungs to their final residence in some portion of the host's body. The second stage larval development proceeds to the adult stage, usually in an organ of the body such as the liver, vena porta or other similar body location. The development of the larvae from the time of entry through the skin to the adult stage takes place in a period of about four to about six weeks. The adult worms form mating pairs and begin immediately to produce eggs which extend the life cycle.
There have been several attempts to isolate extracts having antigenic-immune reaction activity from the various stages of the schistosome life cycle, especially from schistosome eggs (soluble egg antigen-SEA), as well as whole and ground adult worms.
According to one such procedure, live worms are perfused from an infected host, e.g., mice, rat, or rabbit, and are then injected into a test animal. Only minor reductions in worm burden upon challenge with injected cercaria or adult worms is provided by this procedure.
Other studies with antigens derived from adult worms by several different procedures are reported by Shirley E. Maddison, et al., "Studies of Putative Adult Worm-Derived Vaccines and Adjuvants for Protection Against Schistosoma Mansoni Infection in Mice", Journal of Parasitology, Vol. 64, No. 6, pp 986-993 (1978). In one procedure, freshly ground worms (FCW) are prepared by perfusing adult worms from mice with citrate saline at 4.degree. C., collecting the worms in a small amount of Hank's Balanced Salt Solution (HBSS), homogenizing at 4.degree. C. in a tissue grinder, and using the homogenate for injection. In another approach, whole worm extract is prepared from freshly collected adult worms by homogenizing the worms with a small amount of phosphate-buffered saline (PBS) in a tissue grinder. The suspension is clarified by centrifugation and the supernatant fluid is dialyzed against PBS.
With any of these procedures, when test animals which have been injected with the putative worm antigen are challenge-infected with a known number of schistosome cercaria, the percent reduction in the worm burden of the test animals, compared to similar controls, and based on the number of cercaria in the initial infection, ranged from 0-30%.
Greater reductions in the worm burden (up to 52%) are shown by Maddison et al. when the putative antigens from a modified procedure are combined with an injection of a bacteria such as Corynebacterium Parvum.
In the modified procedure, the putative antigen is extracted from adult worms with 0.5 M KCl/1.0 M NaCl. The adult worms are collected at 4.degree. C. and are homogenized in a tissue grinder with a small amount of HBSS in which the worms are collected. Within 30 minutes, the worm homogenate is frozen in dry ice and alcohol at below -100.degree. C. Approximately 5000 of the homogenized worms are extracted with 50 ml of cold extraction medium (0.5 M KCl, 1.0 M NaCl, 2 mM EDTA, 1 mM DL-cysteine HCl in Tris HCl 0.05 M; pH=3.0). The mixture is stirred for 15 minutes at 4.degree. C. and the extract is clarified by centrifugation. The supernatant is held at 4.degree. C. and the solid pellet is re-extracted two times and all the supernatants are pooled and then precipitated by addition of 30% w/v of ammonium sulfate. The suspension is centrifuged (12,000 g for 30 minutes) and the precipitate is redissolved and exhaustively dialyzed against 0.1 M NaCl, 2 mM EDTA, 1 mM DL-cysteine HCl, in Tris HCl 0.05 M; pH=7.6).
In these tests, however, no parallel testing was performed with only the putative antigen without the additional bacteria injections. However, a decreasing concentration of bacteria in the injections (in conjunction with the same amount of putative antigen) shows a decreasing percent reduction in the worm burden. From this, it can be deduced that the primary material responsible for the reduction in the worm burden is the bacteria rather than the putative antigen.
Soluble egg antigen (SEA) from schistosomes are prepared by the techniques shown in the following exemplary publications (as well as references cited therein): Ronald P. Pelley, "Purification of Schistosoma Mansoni Egg Antigens: Theory and Practice," Am. J. Trop. Med. Hyg. 26:104-112 (1977); Fouad N. Boctor, et al., "Isolation of a Polysaccharide Antigen From Schistosoma Mansoni Eggs," J. Immunology, Vol. 122, No. 1, pp 39-43 (January 1979); Clint E. Carter and Daniel G. Colley, "Partial Purification and Characterization of Schistosoma Mansoni Soluble Egg Antigen With Con A-Sepharose Chromatography," J. Immunology, Vol. 122, No. 6, pp 2204-2209 (June 1979); D. Janiece Harrison et al. "Immunoaffinity Purification of Schistosoma Mansoni Soluble Egg Antigens," J. Immunology, Vol. 122, No. 6, pp 2210-2217 (June 1979).
For example, according to the technique of Pelley, about 4,000,000 eggs of Schistosoma mansoni collected from 50 mice are ground in a tissue grinder in 5 ml of 0.5 M NaCl-Con A (concanavilin A) buffer. The suspension is subjected to ultracentrifugation (100,000 g for 2 hours). The supernatant is desalted on Sephadex G-25 (2.times.29 cm column, 0.5 M NaCl--Con A buffer). The desalted supernatant is then subjected to affinity chromatography on Con A Sepharose (1.6.times.21 cm column, 0.5 M NaCl--Con A buffer) eluted with methyl alpha mannose. After concentration of the eluate overnight and dialysis against DEAE buffer, the purified antigen (about 0.2 mg) is obtained by ion exchange chromatography on DEAE cellulose.
The use of blood serum extracted from infected hosts has also met with some success. Serum is collected and concentrated before injection into a test animal. Upon challenge infection by a counted number of cercaria, serum-immunized hosts have shown significant reductions in the worm burden when compared to non-immunized controls.
Nevertheless, still further improvements in the effectiveness in the treatment of and immunization against schistosomiasis and other schistosome induced diseases are required.
Accordingly, it is an object of this invention to provide antigenic material derived from live adult schistosomes which stimulate an immune reaction, thereby protecting an animal host against infection with schistosomes.
Moreover, it is another object of this invention to provide a method of immunizing an animal host against schistosome infection including a method for obtaining antigenic material from live adult schistosomes.
It is a further object of the invention to provide the antigenic material in such pure or nearly pure form as to permit its use as a vaccine in mammals, including humans.